Single cell analysis is a technique for detecting biological molecules in cells on a single cell basis with a high degree of precision and quantifying the molecules. Single cell analysis is roughly composed of two techniques. One is a technique of isolating a cell for analysis, and another is a technique of a sample preparation for measurement in a highly efficient manner from a tiny amount of the biological molecules in one cell and analyzing the sample.
In the former technique, a flow cytometer is generally used. In a flow cytometer, complicated processing is automated, and a user can isolate a cell easily. Meanwhile, a flow cytometer remains expensive. Recently, as a method for isolating a cell more easily and inexpensively, methods using a flow cell device have been known (PTLs 1 and 2).
PTL 1 discloses a configuration in which, for the purpose of isolating a cell of a kind to be measured, plural cell capturing units for individually capturing a cell into a reaction chamber in a flow cell device are disposed in an array form. The cell capturing unit is a device including a membrane filter which can isolate cells introduced into the reaction chamber in the flow cell device one by one. The target cells can not pass through apertures formed in the filter and are captured on the filter and isolated, whereas cells other than the target cells pass through the apertures in the filter and are discharged from the opposite side of the filter. The captured cells are measured by using a fluorescent label introduced by an immunostaining method for staining a membrane protein expressed on a cell membrane, or other methods.
PTL 2 discloses a device and an apparatus for capturing one cell from multiple cells (isolating a cell from multiple cells) and collecting the captured cell. In the case of PTL 2, the cell is captured by suction to a small hole, and thereafter only the target cell is sucked into a reaction chamber by controlling a valve, while discharging other cells than the target cell from the cell capturing unit.
Next, a technique used in a gene expression analysis at a single cell level will be described.
In a previous gene expression analysis realized by a single cell analysis, a method including taking an mRNA from a group of cells, then producing a cDNA which is a complementary strand and subjecting the cDNA to PCR amplification, and further capturing the target to a corresponding probe position using a DNA prove array (DNA chip) to perform fluorescent detection has been used.
However, methods using PCR amplification and a DNA chip have a low precision as a quantification analysis, and a more highly precise method for analyzing a gene expression profile has been demanded. In addition, with the completion of the human genomic analysis, demand for quantitatively investigating gene expressions is increasing, and meanwhile, heterogeneity on a single cell basis in a tissue is attracting attentions. A method for extracting mRNAs from a large number of single cells and quantitatively analyzing the mRNAs is thus demanded.
Recently, a method including subjecting a tiny amount of mRNAs in a single cell to reverse transcription to convert the mRNAs to cDNAs, subjecting the cDNAs to PCR amplification, determining the sequences of the amplification products using a large scale DNA sequencer, and calculating the sequencing results to count the number of the nucleic acid sequences after amplification, thereby estimating the number of the mRNA molecules is beginning to be used (NPL 1). In this method, since the upper limit of the number of measurable genes depends on the number of the parallel provisions of the large scale sequencer, all genes of 20 and several thousand can be measured, and in principle, 100 thousand kinds of sequences including the splicing variant can be measured.
In PTL 3, in order to realize a gene expression analysis of each individual cell at the same time for a large number of cells, a device is disclosed in which a 1st cDNA library is formed using a porous membrane or a membrane device having a large number of beads packed therein, and further a two dimensional distribution of the gene expression is obtained from the library, thereby realizing a gene expression analysis in a large number of cells. In the analysis of gene expression in a single cell using the device, complementary strands (2nd cDNAs) are synthesized from the 1st cDNAs built on the device and subjected to PCR amplification to prepare a sample (sequencing library) for a large scale sequence analyzer.
Since the sequencing library is prepared not for each cell but for each device, there becomes a state where nucleic acids from multiple cells are mixed. In PTL 3, in order to identify a cell on the device from the obtained sequence analysis data, a sequence unique for each position on the device is introduced in the 1st cDNA library, and the results of the sequence analysis obtained by the large scale sequence analyzer are classified for each sequence unique for the position, thereby realizing a gene expression analysis at the single cell level.